Ubiquitylation is one of the most important reversible protein post-translational modifications. Conjugation of the conserved 76 amino acid protein ubiquitin (Ub) mediates diverse biological responses including degradation or subcellular relocalization of the target protein and creation of a docking site for complex formation. In the canonical ubiquitylation pathway, Ub is relayed through three specialized enzymes before establishing an isopeptide linkage between its C-terminus and an acceptor Lys residue on a substrate protein. In the first step, the C-terminus of Ub forms a thiolester conjugate to the active site Cys of the El Ub activating enzyme. In the second step, El-Ub transfers Ub to the active site Cys of an E2 Ub conjugating enzyme. In the final step, an E3 Ub ligase mediates transfer of Ub from E2-Ub to an acceptor Lys of a target protein. Recently, formation of polyUb-modified E2 has been reported to occur in the absence of an E3 and has been attributed solely to the E1 active site. I have discovered that El plus E2 forms polyUb-modified E2 in a manner that cannot be explained by the E1 active site. Specifically, reactions of El with Ubc4 produced Lys48-linked polyUb chains, whereas reactions of El with Ubc13/Mms2 produced Lys63-linked polyUb chains, characteristic of Ubc13/Mms2 reactions. I hypothesize that previously unappreciated features of E2 Ub conjugating enzymes are responsible for E3-independent polyUb chain formation. As a predoctoral fellow, I propose experiments to uncover the features and the mechanism of E3-independent polyUb chain formation and to determine its physiological significance using a combination of site-directed mutagenesis, mass spectrometry, gene replacement and phenotypic analysis. These experiments are fundamentally important because they call into question the catalytic roles of El, E2 and E3 enzymes and have strong potential to redefine the current understanding of ubiquitylation. Ubiquitylation has roles in almost all known biological processes from development and cell cycle regulation to programmed cell death. Mutations in multiple components of the E1, E2 and E3 system have been discovered in human disease. By clarifying the biochemical basis for polyUb formation by E2, 1 aim to elucidate how E2 enzymes are dynamically regulated in multiple aspects of human health.